CN111175495A - Kit for determining content of gastrin17 and using method thereof - Google Patents
Kit for determining content of gastrin17 and using method thereof Download PDFInfo
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- CN111175495A CN111175495A CN202010124959.1A CN202010124959A CN111175495A CN 111175495 A CN111175495 A CN 111175495A CN 202010124959 A CN202010124959 A CN 202010124959A CN 111175495 A CN111175495 A CN 111175495A
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- 108010066264 gastrin 17 Proteins 0.000 title claims abstract description 76
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kit for measuring the content of gastrin17 and a using method thereof in the related technical field of biochemistry, comprising a calibrator, a quality control material, an anti-reagent, a magnetic particle reagent and a luminescent substrate; wherein the anti-reagent is fluorescein isothiocyanate labeled gastrin17 coating antibody and alkaline phosphatase labeled gastrin17 labeled antibody; the magnetic particle reagent is a magnetic particle and goat anti-FITC conjugate; the invention combines the chemiluminescence technology with the immunomagnetic particles, provides a reaction system close to homogeneous phase, adopts a one-step reaction mode, greatly improves the detection sensitivity and the precision, enlarges the detection range, greatly shortens the reaction time, and is less than 35min from the beginning of sample application to the detection result, which is obviously faster than similar kits; and a plurality of samples can be simultaneously measured on a full-automatic chemiluminescence apparatus, high-flux rapid measurement of the gastrin17 is realized, the accuracy is high, the specificity is strong, and the accuracy and the detection efficiency are greatly improved.
Description
Technical Field
The invention relates to the technical field of biochemistry correlation, in particular to a kit for measuring the content of gastrin17 and a using method thereof.
Background
Gastrin17 (gastrin17, G17) is an important gastrointestinal peptide hormone, first discovered and named by Edkins, a british scholar in 1905. It is secreted mainly by G cells. G cells are typically open cells, most frequently in the antrum, and secondarily in the fundus, duodenum, and jejunum. The D cells of human islets also secrete gastrin.
Gastrin17 stimulates gastric acid secretion, promotes cell proliferation and inhibits apoptosis, and also has effects on cancer cell growth and malignant transformation of cells. Meanwhile, gastrin can promote secretion of pepsin, secretin, pancreatic juice and bile in gastrointestinal tracts, promote release of calcitonin and insulin, and strengthen gastrointestinal tract movement and gallbladder contraction. In recent years, serological biopsy indexes of gastric mucosa such as serum gastrin17, pepsinogen I/II, helicobacter pylori (H.pyrori) antibodies and the like are widely regarded in clinical auxiliary diagnosis of gastrointestinal diseases by virtue of the characteristics of low cost, simplicity, convenience, non-invasiveness, dynamic follow-up and the like.
Currently known methods for measuring gastrin17 include Fluorescence Immunochromatography (FICA), enzyme-linked immunosorbent assay (ELISA), chemiluminescence, and the like. The organic dye marker of the fluorescence immunochromatography is easy to decompose under illumination, is easy to generate a photobleaching phenomenon, has a concentration quenching characteristic, and can influence the accuracy and reliability of an analysis result. The ELISA has the defects of long detection time, complex operation and poor repeatability, and is not suitable for the needs of emergency treatment and timely diagnosis of clinical patients.
Disclosure of Invention
Aiming at the technical problems of long detection time, complex operation, poor repeatability and unsuitability for emergency treatment and timely diagnosis of clinical patients in the prior art, the invention provides a kit for measuring the content of gastrin17 and a using method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a kit for determining the content of gastrin17 comprises a calibrator, a quality control material, an anti-reagent, a magnetic particle reagent and a luminescent substrate; the calibrator, the quality control product, the anti-reagent, the magnetic particle reagent and the luminescent substrate are respectively and independently packaged;
the calibrator is prepared by adding 0.01-0.05 g of tetracycline and 0.1-0.5 g of neomycin sulfate into 1L of newborn calf serum, completely dissolving the tetracycline and the neomycin sulfate, and then treating the mixture with a 0.22 mu m filter membrane;
the quality control product is prepared by dissolving gastrin17 antigen by the calibrator;
the anti-reagent is prepared by the following steps:
sa 1: mixing 10-20 g of Na2PO3H·12H2O, 1-2 g of NaPO3H2·12H2Adding O, 1-5 g of sheep serum, 3-10 g of newborn bovine serum and 1-5 g of horse serum into 1L of purified water, fully stirring until the mixture is completely dissolved, and adjusting the pH value to 5-6 by using 4M HCl to prepare an anti-reagent buffer solution;
sa 2: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 1.0-5.0 mg/mL by using the anti-reagent buffer solution, adding the fluorescein isothiocyanate solution with the mass 1.1-1.5 times that of the gastrin17 antibody into the gastrin17 antibody, fully mixing, balancing by using a carbonate buffer solution with the pH value of 8-9, and then separating and purifying by using gel chromatography to prepare the fluorescein isothiocyanate labeled gastrin17 coated antibody:
sa 3: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 1.0-5.0 mg/mL by using the anti-reagent buffer solution, respectively activating reactive groups by using a gastrin17 antibody and the alkaline phosphatase, and then performing alkaline phosphatase treatment according to the molar ratio: the method comprises the following steps of (1) fully and uniformly mixing a gastrin17 antibody in a reaction ratio of 1: 1-1: 3 under the catalysis of a catalyst to perform coupling reaction, balancing the antibody with a Tris buffer solution with the pH value of 8-9 after the sufficient reaction is performed, and performing separation and purification on gel columns with different molecular sizes to prepare an alkaline phosphatase-labeled gastrin17 labeled antibody;
sa 4: adding the fluorescein isothiocyanate labeled gastrin17 coating antibody and the alkaline phosphatase labeled gastrin17 labeled antibody into a phosphate buffer solution containing a surfactant, and fully mixing to obtain the compound;
the magnetic particle reagent is prepared by coupling an anti-fluorescein isothiocyanate antibody and a carboxyl magnetic bead;
the luminogenic substrate is prepared by dissolving ALPS in a luminogenic substrate buffer.
The cleaning solution is prepared by dissolving 150-170 g of NaCl, 3-5 g of KCl, 24-24.4 g of tris (hydroxymethyl) aminomethane and 0.8-1.2 mL of Tween20 in 900mL of double distilled water, adjusting the pH value to 7.2-7.6 by using HCl, and finally fixing the volume to 1000mL by using the double distilled water.
Preferably, the surfactant in Sa4 is one or more of Tween20, TritonX-100 and Bronidox, and the addition amount of the surfactant is 0.01-0.5%.
Further, the magnetic particle reagent is prepared by the following operation:
sb 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 15-20 min, absorbing a supernatant after all the carboxyl magnetic beads are settled, adding a magnetic particle buffer solution with the volume 2-5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 20-30 min, placing in the magnetic field for 15-20 min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 1-3 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 10-50 mg/mL to prepare a carboxyl magnetic bead solution;
sb 2: adding an anti-fluorescein isothiocyanate antibody accounting for 0.8-1.2% of the mass of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 16-20 hours in a uniformly mixed state at 2-8 ℃;
sb 3: and (3) placing the solution prepared from Sb2 in a magnetic field for 12-18 min, washing the magnetic beads for 1-3 times by using a magnetic particle buffer solution after the carboxyl magnetic beads are settled, then fixing the volume to 8-12 mg/mL, and storing at 2-8 ℃.
Further, the magnetic particle buffer solution is prepared by adding 12.12-15.26 mg of Tris, 5.82-8.58 mg of NaCl and 50-60 g of methyl cellulose ether into 1L of purified water, and fully stirring until the mixture is completely dissolved.
Further, the luminescent substrate is prepared by fully dissolving ALPS in a luminescent substrate buffer solution with the volume of 4-10 times that of ALPS; the luminescent substrate buffer solution is prepared by adding 12.12-121.14 g of Tris, 5.82-8.58 g of NaCl and 0.03-0.05 g of lucigenin into 1L of purified water, fully stirring until the components are completely dissolved, and adjusting the pH value to 9.3-9.5 by hydrochloric acid.
A method of using the kit for determining the amount of gastrin17 as described in any one of the above methods, comprising the steps of:
sc 1: taking three test tubes, and respectively adding 100 mu L of the calibrator, 100 mu L of the quality control material and 100 mu L of a sample to be detected;
sc 2: adding 60 mu L of the anti-reagent into each test tube, covering the test tube with a plastic film, slightly shaking for 30s, and placing in a water bath at 37 ℃ for 15 min;
sc 3: adding 30 mu L of the magnetic particle reagent into each test tube, covering the test tubes with the plastic film, slightly oscillating for 30s, and placing in a water bath at 37 ℃ for 5 min;
sc 4: precipitating the test tube on a magnetic separator for 3min, slowly inverting the test tube and the magnetic separator, and pouring out the supernatant; placing the inverted test tube together with the magnetic separator on a filter paper, and patting the bottom of the magnetic separator to remove any droplets adhering to the wall of the test tube;
sc 5: and adding 200 mu L of the luminescent substrate into each test tube, oscillating and uniformly mixing for 3s, and detecting the luminous intensity by using a chemiluminescence apparatus.
Further, the Sc4 operation also includes sc4.1: adding 300. mu.L of the washing solution to each of the test tubes, covering the test tube with a plastic film, gently shaking the test tube for 30s, slowly inverting the test tube and the magnetic separator after mixing, pouring out the supernatant, placing the inverted test tube together with the magnetic separator on a filter paper, and strongly tapping the bottom of the separator to remove any droplets stuck to the tube wall.
Further, after the operation of sc4.1, the method also includes sc4.2: repeating the operation of Sc4.2 for 1-2 times.
The invention has the following advantages:
1. the kit combines a chemiluminescence technology with immunomagnetic particles, provides a reaction system close to homogeneous phase, adopts a one-step reaction mode, greatly improves the detection sensitivity and precision, enlarges the detection range, greatly shortens the reaction time, and is less than 35min from the beginning of sample application to the detection result, which is obviously faster than similar kits;
2. the invention discloses a novel FITC antibody and magnetic particle coupling method which has the advantages of high coupling efficiency, firm combination and stable process, and greatly reduces the product cost while improving the product performance;
3. the anti-reagent, the magnetic particle reagent, the calibrator, the quality control product, the luminous substrate solution and the concentrated washing solution in the kit are all optimal formulas under the reaction system, and powerful guarantee is provided for the service life and the detection performance of the kit;
4. the method can be used for simultaneously measuring a plurality of samples on a full-automatic chemiluminescence apparatus, realizes high-flux rapid measurement of the gastrin17, and has the advantages of high accuracy, strong specificity and greatly improved accuracy and detection efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the correlation of the kit of the present invention with other commercially available kits for detecting clinical sera; wherein the abscissa is the detection result of other commercially available kits, and the ordinate is the detection result of the kit of the invention.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. Moreover, in the following description, descriptions of well-known structures, techniques, and operations are omitted so as to not unnecessarily obscure the concepts of the present invention. In addition, the technical features related to the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In the present invention, the proportion and content of the unit are not particularly specified, the solid component is the mass proportion and content, and the liquid component is the volume proportion and content.
A kit for measuring the content of gastrin17 comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and a cleaning solution; wherein, the calibrator, the quality control product, the anti-reagent, the magnetic particle reagent, the luminescent substrate and the cleaning solution are respectively and independently packaged to form a kit for measuring the content of the gastrin17, and the specific embodiment is as follows:
example 1
The calibrator is prepared by adding 0.01g of tetracycline and 0.1g of neomycin sulfate into 1L of newborn calf serum, and performing 0.22 mu m filter membrane treatment after complete dissolution;
the quality control product is prepared by dissolving gastrin17 antigen with calibrator;
the anti-reagent is prepared by the following steps:
sa 1: adding 10g of Na2PO3H & 12H2O, 1g of NaPO3H2 & 12H2O, 1g of sheep serum, 3g of newborn bovine serum and 1g of horse serum into 1L of purified water, fully stirring until the mixture is completely dissolved, and adjusting the pH to 5 by using 4M HCl to prepare an anti-reagent buffer solution;
sa 2: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 1.0mg/mL by using an anti-reagent buffer solution, adding the fluorescein isothiocyanate solution with the mass being 1.1 times that of the gastrin17 antibody into the gastrin17 antibody, fully mixing, balancing by using a carbonate buffer solution with the pH value of 8, and then separating and purifying by using gel chromatography to prepare the fluorescein isothiocyanate labeled gastrin17 coated antibody:
sa 3: alkaline phosphatase was prepared in an alkaline phosphatase solution with an anti-reagent buffer solution at a concentration of 1.0mg/mL, and the reaction groups were activated with a gastrin17 antibody and alkaline phosphatase, respectively, at a molar ratio of alkaline phosphatase: the gastrin17 antibody is in a reaction ratio of 1:1, the coupling reaction is carried out by fully and uniformly mixing under the catalysis of a catalyst, after the full reaction, Tris buffer solution with pH of 8 is used for balancing, and a gel column is used for separating and purifying different molecular sizes to prepare the gastrin17 labeled antibody labeled by alkaline phosphatase;
sa 4: adding a fluorescein isothiocyanate labeled gastrin17 coating antibody and an alkaline phosphatase labeled gastrin17 labeled antibody into a phosphate buffer solution containing a surfactant, and fully mixing to obtain the composition;
wherein the surfactant is Tween20, and the addition amount of the surfactant is 0.01%;
the magnetic particle reagent is prepared by the following steps:
sb 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 15min, absorbing the supernatant after the carboxyl magnetic beads are completely settled, adding a magnetic particle buffer solution with the volume 2 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 20min, placing in the magnetic field for 15min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 1 time; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 10mg/mL to prepare a carboxyl magnetic bead solution;
sb 2: adding an anti-fluorescein isothiocyanate antibody accounting for 0.8 percent of the mass of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and keeping the mixture in a uniformly mixed state at the temperature of 2 ℃ for reacting for 16 hours;
sb 3: placing the solution prepared from Sb2 in a magnetic field for 12min, washing for 1 time by using magnetic particle buffer solution after the carboxyl magnetic beads are settled, then fixing the volume to 8mg/mL, and storing at 2 ℃;
wherein, the magnetic particle buffer solution is prepared by adding 12.12mg of Tris, 5.82mg of NaCl and 50g of methyl cellulose ether into 1L of purified water and fully stirring until the Tris, the NaCl and the methyl cellulose ether are completely dissolved;
the luminescent substrate was prepared by dissolving ALPS sufficiently in a luminescent substrate buffer 4 times the volume of ALPS; the luminescent substrate buffer solution is prepared by adding 12.12g of Tris, 5.82g of NaCl and 0.03g of lucigenin into 1L of purified water, fully stirring until the Tris and the NaCl are completely dissolved, and adjusting the pH value to 9.3 by hydrochloric acid;
the cleaning solution is prepared by dissolving 150g NaCl, 3g KCl, 24g trihydroxymethyl aminomethane and 0.8mL Tween20 in 900mL double distilled water, adjusting pH to 7.2 with HCl, and fixing volume to 1000mL with double distilled water.
Example 2
The calibrator is prepared by adding 0.03g of tetracycline and 0.3g of neomycin sulfate into 1L of newborn calf serum, and performing 0.22 mu m filter membrane treatment after complete dissolution;
the quality control product is prepared by dissolving gastrin17 antigen with calibrator;
the anti-reagent is prepared by the following steps:
sa 1: adding 15g of Na2PO3H & 12H2O, 1.5g of NaPO3H2 & 12H2O, 3g of sheep serum, 6g of newborn bovine serum and 3g of horse serum into 1L of purified water, fully stirring until the mixture is completely dissolved, and adjusting the pH to 5.5 by using 4M HCl to prepare an anti-reagent buffer solution;
sa 2: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 3.0mg/mL by using an anti-reagent buffer solution, adding the fluorescein isothiocyanate solution with the mass being 1.3 times that of the gastrin17 antibody into the gastrin17 antibody, fully mixing, balancing by using a carbonate buffer solution with the pH value of 8.5, and then separating and purifying by using gel chromatography to prepare the fluorescein isothiocyanate labeled gastrin17 coated antibody:
sa 3: alkaline phosphatase was prepared in an anti-reagent buffer solution at a concentration of 3.0mg/mL, and the molar ratio of alkaline phosphatase after activation of the reactive group with the gastrin17 antibody and alkaline phosphatase, respectively, was: the gastrin17 antibody is in a reaction ratio of 1:2, the coupling reaction is carried out by fully and uniformly mixing under the catalysis of a catalyst, after the full reaction, Tris buffer solution with pH of 8.5 is used for balancing, and a gel column is used for separating and purifying different molecular sizes to prepare the gastrin17 labeled antibody labeled by alkaline phosphatase;
sa 4: adding a fluorescein isothiocyanate labeled gastrin17 coating antibody and an alkaline phosphatase labeled gastrin17 labeled antibody into a phosphate buffer solution containing a surfactant, and fully mixing to obtain the composition;
wherein the surfactant is TritonX-100, and the addition amount of the surfactant is 0.3%;
the magnetic particle reagent is prepared by the following steps:
sb 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 18min, absorbing the supernatant after the carboxyl magnetic beads are completely settled, adding a magnetic particle buffer solution with the volume 4 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 25min, placing in the magnetic field for 18min, and absorbing the supernatant; repeatedly washing the carboxyl magnetic beads for 2 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 30mg/mL to prepare a carboxyl magnetic bead solution;
sb 2: adding an anti-fluorescein isothiocyanate antibody accounting for 1 percent of the mass of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 18 hours in a uniformly mixed state at 5 ℃;
sb 3: placing the solution prepared from Sb2 in a magnetic field for 15min, washing for 2 times by using magnetic particle buffer solution after the carboxyl magnetic beads are settled, then fixing the volume to 10mg/mL, and storing at 5 ℃;
wherein, the magnetic particle buffer solution is prepared by adding 14.26mg of Tris, 6.58mg of NaCl and 55g of methyl cellulose ether into 1L of purified water and fully stirring until the Tris and the NaCl are completely dissolved;
the luminescent substrate was prepared by dissolving ALPS sufficiently in a luminescent substrate buffer solution 7 times the volume of ALPS; the luminescent substrate buffer solution is prepared by adding 40.14g of Tris, 6.58g of NaCl and 0.04g of lucigenin into 1L of purified water, fully stirring until the Tris and the NaCl are completely dissolved, and adjusting the pH value to 9.4 by hydrochloric acid;
the cleaning solution is prepared by dissolving 160g NaCl, 4g KCl, 24.2g trihydroxymethyl aminomethane and 1mL Tween20 in 900mL double distilled water, adjusting pH to 7.4 with HCl, and fixing volume to 1000mL with double distilled water.
Example 3
The calibrator is prepared by adding 0.05g of tetracycline and 0.5g of neomycin sulfate into 1L of newborn calf serum, and performing 0.22 mu m filter membrane treatment after complete dissolution;
the quality control product is prepared by dissolving gastrin17 antigen with calibrator;
the anti-reagent is prepared by the following steps:
sa 1: adding 20g of Na2PO3H & 12H2O, 2g of NaPO3H2 & 12H2O, 5g of sheep serum, 10g of newborn bovine serum and 5g of horse serum into 1L of purified water, fully stirring until the sheep serum is completely dissolved, and adjusting the pH to 6 by using 4M HCl to prepare an anti-reagent buffer solution;
sa 2: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 5.0mg/mL by using an anti-reagent buffer solution, adding the fluorescein isothiocyanate solution with the mass being 1.5 times that of the gastrin17 antibody into the gastrin17 antibody, fully mixing, balancing by using a carbonate buffer solution with the pH value of 9, and then separating and purifying by using gel chromatography to prepare the fluorescein isothiocyanate labeled gastrin17 coated antibody:
sa 3: alkaline phosphatase was prepared in an alkaline phosphatase solution at a concentration of 5.0mg/mL using an anti-reagent buffer, and the reaction groups were activated with a gastrin17 antibody and alkaline phosphatase, respectively, at a molar ratio of alkaline phosphatase: the gastrin17 antibody is in a reaction ratio of 1:3, is fully and uniformly mixed under the catalysis of a catalyst for coupling reaction, is balanced by using a Tris buffer solution with the pH value of 9 after full reaction, and is separated and purified by a gel column with different molecular sizes to prepare the gastrin17 labeled antibody labeled by alkaline phosphatase;
sa 4: adding a fluorescein isothiocyanate labeled gastrin17 coating antibody and an alkaline phosphatase labeled gastrin17 labeled antibody into a phosphate buffer solution containing a surfactant, and fully mixing to obtain the composition;
wherein the surfactant is Bronidox, and the addition amount of the surfactant is 0.5%;
the magnetic particle reagent is prepared by the following steps:
sb 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 20min, absorbing the supernatant after the carboxyl magnetic beads are completely settled, adding a magnetic particle buffer solution with the volume 5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 30min, placing in the magnetic field for 20min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 3 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 50mg/mL to prepare a carboxyl magnetic bead solution;
sb 2: adding an anti-fluorescein isothiocyanate antibody accounting for 1.2 percent of the mass of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 20 hours in a uniformly mixed state at the temperature of 8 ℃;
sb 3: placing the solution prepared from Sb2 in a magnetic field for 18min, washing for 3 times by using magnetic particle buffer solution after the carboxyl magnetic beads are settled, then fixing the volume to 12mg/mL, and storing at 8 ℃;
wherein, the magnetic particle buffer solution is prepared by adding 15.26mg of Tris, 8.58mg of NaCl and 60g of methyl cellulose ether into 1L of purified water and fully stirring until the Tris and the NaCl are completely dissolved;
the luminescent substrate is prepared by dissolving ALPS in a luminescent substrate buffer solution in a volume 10 times that of ALPS; the luminescent substrate buffer solution is prepared by adding 121.14g of Tris, 8.58g of NaCl and 0.05g of lucigenin into 1L of purified water, fully stirring until the Tris and the NaCl are completely dissolved, and adjusting the pH value to 9.5 by hydrochloric acid;
the cleaning solution is prepared by dissolving 170g NaCl, 5g KCl, 24.4g trihydroxymethyl aminomethane and 1.2mL Tween20 in 900mL double distilled water, adjusting pH to 7.6 with HCl, and fixing volume to 1000mL with double distilled water.
The specific operation of using the kit for measuring the content of gastrin17 prepared in any one of embodiments 1 to 3 to measure the content of gastrin17 is as follows:
1. sample collection
Serum was collected using the correct medical technique (severely hemolyzed or lipemic specimens could not be used for the assay), and the collected specimens were allowed to stand at room temperature for no more than 8 hours; if the detection is not carried out within 8 hours, the sample needs to be placed in a refrigerator at the temperature of 2-8 ℃; if the food needs to be stored or transported for more than 72 hours, the food should be frozen below-20 ℃ to avoid repeated freezing and thawing. Before use, return to room temperature and mix by gentle shaking.
2. Preparation before experiment
2.1 taking a bottle of washing liquor to dilute by 15 times by using distilled water;
2.2, adjusting the temperature of the constant temperature box or the water bath kettle to 37 ℃, and using the constant temperature box or the water bath kettle after the temperature is stable;
2.3 mix the magnetic particle suspension well until no visible precipitation.
3 Experimental methods
Sc 1: taking three test tubes and numbering; respectively adding 100 mu L of calibrator, 100 mu L of quality control material and 100 mu L of sample to be detected;
sc 2: adding 60 μ L of the anti-reagent into each test tube, covering the test tube with a plastic film, slightly oscillating for 30s, and placing in a water bath at 37 ℃ for 15 min;
sc 3: adding 30 μ L magnetic particle reagent into each test tube, covering the test tube with plastic film, gently oscillating for 30s, and placing in water bath at 37 deg.C for 5 min;
sc 4: precipitating the test tube on a magnetic separator for 3min, slowly inverting the test tube and the magnetic separator, and pouring out the supernatant; placing the inverted test tube together with the magnetic separator on a filter paper and slapping the bottom of the magnetic separator to remove any droplets adhering to the wall of the tube;
sc4.1: adding 300 μ L of cleaning solution into each test tube, covering the test tube with plastic film, slightly shaking the test tube for 30s, slowly inverting the test tube and magnetic separator after mixing, pouring out supernatant, putting the inverted test tube and magnetic separator together on filter paper, and strongly slapping the bottom of the separator to remove all droplets adhered to the tube wall; in order to improve the accuracy of the detection data, the steps can be repeated for 1-2 times during specific implementation;
sc 5: adding 200 μ L luminescent substrate into each test tube, shaking and mixing for 3s, and detecting the luminescence intensity with chemiluminescence apparatus.
And establishing a calibration curve by adopting a four-parameter fitting mode and taking the concentration value of the calibrator as an X axis and the luminous intensity value of the calibrator as a Y axis. And calculating back the corresponding concentration value according to the luminous intensity value of the sample to be detected.
The kit disclosed by the invention can be identified according to methodology to achieve the following indexes:
the standard curve is linear: r is more than 0.9900;
the lowest detection limit is: less than or equal to 0.5 ng/ml;
accuracy: the adding recovery rate is 85-115%;
repeatability: coefficient of variation CV is less than or equal to 8 percent;
inter-batch difference: the coefficient of variation is less than or equal to 15 percent;
linear dilution: r is greater than 0.9900.
The correlation between the detection results of the kit of the present invention and the commercial gastrin17 kit was compared by comparing the measurement values of 100 serum samples with those of the commercial gastrin17 kit a, and the results are shown in fig. 1.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments of the invention described above, and that equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A kit for determining the amount of gastrin17, comprising: comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent and a luminescent substrate; the calibrator, the quality control product, the anti-reagent, the magnetic particle reagent and the luminescent substrate are respectively and independently packaged;
the calibrator is prepared by adding 0.01-0.05 g of tetracycline and 0.1-0.5 g of neomycin sulfate into 1L of newborn calf serum, completely dissolving the tetracycline and the neomycin sulfate, and then treating the mixture with a filter membrane;
the quality control product is prepared by dissolving gastrin17 antigen by the calibrator;
the anti-reagent is prepared by the following steps:
sa 1: mixing 10-20 g of Na2PO3H·12H2O, 1-2 g of NaPO3H2·12H2Adding O, 1-5 g of sheep serum, 3-10 g of newborn bovine serum and 1-5 g of horse serum into 1L of purified water, fully stirring until the mixture is completely dissolved, and adjusting the pH value to 5-6 by using HCl to prepare an anti-reagent buffer solution;
sa 2: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 1.0-5.0 mg/mL by using the anti-reagent buffer solution, adding the fluorescein isothiocyanate solution with the mass 1.1-1.5 times that of the gastrin17 antibody into the gastrin17 antibody, fully mixing, balancing by using a carbonate buffer solution with the pH value of 8-9, and then separating and purifying by using gel chromatography to prepare the fluorescein isothiocyanate labeled gastrin17 coated antibody:
sa 3: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 1.0-5.0 mg/mL by using the anti-reagent buffer solution, respectively activating reactive groups by using a gastrin17 antibody and the alkaline phosphatase, and then performing alkaline phosphatase treatment according to the molar ratio: the method comprises the following steps of (1) fully and uniformly mixing a gastrin17 antibody in a reaction ratio of 1: 1-1: 3 under the catalysis of a catalyst to perform coupling reaction, balancing by using a Tris buffer solution after the full reaction, and performing separation and purification on gel columns with different molecular sizes to prepare an alkaline phosphatase-labeled gastrin17 labeled antibody;
sa 4: adding the fluorescein isothiocyanate labeled gastrin17 coating antibody and the alkaline phosphatase labeled gastrin17 labeled antibody into a phosphate buffer solution containing a surfactant, and fully mixing to obtain the compound;
the magnetic particle reagent is prepared by coupling an anti-fluorescein isothiocyanate antibody and a carboxyl magnetic bead;
the luminogenic substrate is prepared by dissolving ALPS in a luminogenic substrate buffer.
2. The kit for determining the amount of gastrin17 according to claim 1, wherein: the cleaning solution is prepared by dissolving 150-170 g of NaCl, 3-5 g of KCl, 24-24.4 g of tris (hydroxymethyl) aminomethane and 0.8-1.2 mL of Tween20 in 900mL of double distilled water, adjusting the pH value to 7.2-7.6 by using HCl, and finally fixing the volume to 1000mL by using the double distilled water.
3. The kit for determining the amount of gastrin17 according to claim 1 or 2, wherein: in Sa4, the surfactant is one or more of Tween20, TritonX-100 and Bronidox, and the addition amount of the surfactant is 0.01-0.5%.
4. The kit for determining the amount of gastrin17 according to claim 1 or 2, wherein: the magnetic particle reagent is prepared by the following steps:
sb 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 15-20 min, absorbing a supernatant after all the carboxyl magnetic beads are settled, adding a magnetic particle buffer solution with the volume 2-5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 20-30 min, placing in the magnetic field for 15-20 min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 1-3 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 10-50 mg/mL to prepare a carboxyl magnetic bead solution;
sb 2: adding an anti-fluorescein isothiocyanate antibody accounting for 0.8-1.2% of the mass of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 16-20 hours in a uniformly mixed state at 2-8 ℃;
sb 3: and (3) placing the solution prepared from Sb2 in a magnetic field for 12-18 min, washing the magnetic beads for 1-3 times by using a magnetic particle buffer solution after the carboxyl magnetic beads are settled, then fixing the volume to 8-12 mg/mL, and storing at 2-8 ℃.
5. The kit for determining the amount of gastrin17 according to claim 4, wherein: the magnetic particle buffer solution is prepared by adding 12.12-15.26 mg of Tris, 5.82-8.58 mg of NaCl and 50-60 g of methyl cellulose ether into 1L of purified water, and fully stirring until the Tris, the NaCl and the methyl cellulose ether are completely dissolved.
6. The kit for determining the amount of gastrin17 according to claim 1 or 2, wherein: the luminescent substrate is prepared by fully dissolving luminescent substrate buffer solution with the volume 4-10 times of that of ALPS in ALPS; the luminescent substrate buffer solution is prepared by adding 12.12-121.14 g of Tris, 5.82-8.58 g of NaCl and 0.03-0.05 g of lucigenin into 1L of purified water, fully stirring until the components are completely dissolved, and adjusting the pH value to 9.3-9.5 by hydrochloric acid.
7. The use method of the kit for determining the content of gastrin17 according to any one of claims 1 to 6, is characterized in that: the method comprises the following steps:
sc 1: taking three test tubes, and respectively adding 100 mu L of the calibrator, 100 mu L of the quality control material and 100 mu L of a sample to be detected;
sc 2: adding 60 mu L of the anti-reagent into each test tube, covering the test tube with a plastic film, slightly shaking for 30s, and placing in a water bath at 37 ℃ for 15 min;
sc 3: adding 30 mu L of the magnetic particle reagent into each test tube, covering the test tubes with the plastic film, slightly oscillating for 30s, and placing in a water bath at 37 ℃ for 5 min;
sc 4: precipitating the test tube on a magnetic separator for 3min, slowly inverting the test tube and the magnetic separator, and pouring out the supernatant; placing the inverted test tube together with the magnetic separator on a filter paper, and patting the bottom of the magnetic separator to remove any droplets adhering to the wall of the test tube;
sc 5: and adding 200 mu L of the luminescent substrate into each test tube, oscillating and uniformly mixing for 3s, and detecting the luminous intensity by using a chemiluminescence apparatus.
8. The method for using the kit for determining the content of gastrin17 according to claim 7, wherein: after Sc4 operation, Sc4.1: adding 300. mu.L of washing solution to each of the test tubes, covering the test tube with a plastic film, gently shaking the test tube for 30s, slowly inverting the test tube and the magnetic separator after mixing, pouring out the supernatant, placing the inverted test tube together with the magnetic separator on a filter paper, and vigorously patting the bottom of the separator to remove any droplets adhering to the tube wall.
9. The method for using the kit for determining the content of gastrin17 according to claim 8, wherein: after the operation of sc4.1, the operation also includes sc4.2: repeating the operation of Sc4.2 for 1-2 times.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104614536A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting gastrin-17 and preparation method as well as application thereof |
| CN104914251A (en) * | 2015-05-22 | 2015-09-16 | 必欧瀚生物技术（合肥）有限公司 | Gastrin-17 enzymatic chemiluminescence immunoassay kit |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
| US20180231540A1 (en) * | 2015-02-10 | 2018-08-16 | Shenzhen New Industries Biomedical Engineering Co., Ltd. | Reagent kit used for detecting gastrin-17, and preparation method and application for reagent kit |
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2020
- 2020-02-27 CN CN202010124959.1A patent/CN111175495A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104614536A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting gastrin-17 and preparation method as well as application thereof |
| US20180231540A1 (en) * | 2015-02-10 | 2018-08-16 | Shenzhen New Industries Biomedical Engineering Co., Ltd. | Reagent kit used for detecting gastrin-17, and preparation method and application for reagent kit |
| CN104914251A (en) * | 2015-05-22 | 2015-09-16 | 必欧瀚生物技术（合肥）有限公司 | Gastrin-17 enzymatic chemiluminescence immunoassay kit |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
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